State of the art fluorescence measurement techniques enable detection and analysis of samples of one or more substances in a contact-free manner. Various known fluorescence measurement techniques include labeling the substances with one or more selected fluorescent dyes, or employing the intrinsic fluorescence of the substances. For analyses of this type, samples of the substances are illuminated with exciting radiation or light of wavelengths or wavelength regions which are suited to the absorption behavior of the intrinsic fluorescence or the fluorescent dyes used (usually the absorption maxima). The response of the intrinsic fluorescence or fluorescent dyes is to emit light characteristic for the intrinsic fluorescence or fluorescent dyes, whose wavelength is usually longer than that of the exciting radiation or light. Hence, it is possible to measure quantitatively changes in the physical property, for example concentration of the substance in a solution, composition, physical environment, and similar parameters, via changes in the fluorescence behavior, for example changes in the fluorescence intensity and/or in the wavelength of the absorption and/or emission.
In one particular known class of fluorescence measurement or analyses called adherent cell assays, adherent cells of various tissue types are grown in culture and incubated in a growth medium. A first known subclass of adherent cell assays requires that the growth medium include a fluorescent dye. The cells will absorb the dye at particular rates, and these rates may be correlated with various physiological functions of the cells such as K+ channel activity. A cell which has absorbed dye will typically fluoresce at an enhanced intensity as compared to the growth medium which incorporates that dye. Fluorescence measurements or analyses of this type are of significant importance in the pharmaceutical industry since they may be employed to screen a variety of tissue types for interaction with chemical species of pharmaceutical interest. In an analysis of this type, adherent cells may be cultured in a tissue culture dish, or alternatively in a tissue culture treated microtiter plate which includes a plurality of wells.
FIGS. 1A and 1B show an example of a known type of 96 well microtiter plate or carrier 130. In this case, each of the microtiter plate wells has a substantially planar, optically transparent bottom wall or member 132, such as a plastic film or a quartz plate. Tissue cells are cultured in a layer on the upper surface of the bottom member with a supernatant layer of growth medium thereabove. Chemical species being assayed are placed into the supernatant liquid together with a fluorescent dye. Then, the effect of the chemical species on cell metabolism is assayed by measuring the fluorescence of the cell layers. Such techniques are well known in the art and are described, for example, in U.S. Pat. Nos. 4,343,782, 4,835,103 and PCT published application WO 90/15317. In order to measure the fluorescence of the cells, the cell layers are illuminated with light of a first wavelength and emission at a second wavelength is monitored by a photodetector device, for example a camera. Problems may arise in this type of an assay because the cell layer is typically on the order of 10 microns in thickness, while the depth of the supernatant liquid is on the order of many millimeters. While the relative intensity of the emission from the supernatant liquid is generally lower than that from the cells which have absorbed the dye, fluorescence from the supernatant liquid may constitute a significant source of error in these assays because of the large relative volume of the supernatant in the fluorescence detection volume.
A second known subclass of adherent cell assays is like the first subclass except that instead of the supernatant layer of growth medium including a fluorescent dye, the cells are intrinsically fluorescent by virtue of their expression of a fluorescent genetic reporter such as green fluorescent protein. Although the supernatant layer in this case typically does not contain a fluorescent dye, the autofluorescence of the supernatant layer may constitute a significant source of error in these assays because of the large relative volume of the supernatant in the fluorescence detection volume.
In another particular known class of fluorescence analyses called homogeneous fluorescence assays, all the components of the assay are present during measurement. The reactions occur in solution generally without a solid-phase attachment. Problems may arise in this type of an assay because of variability in the contents among a plurality of containers of the solution, for example the plurality of wells in a microtiter plate, wherein the variable factors may include the total volume of solution in each well, attenuation of both the excitation and emission radiation in the solution, and surface tension, all potentially affecting the intensity and wavelengths of the volume-integrated fluorescence measured in each container.
A known subclass of homogeneous fluorescence assays comprises fluorescence polarization assays, which involve polarization sensitive detection of fluorescent emission in response to polarized excitation. In addition to the problems related to homogeneous fluorescence assays described above, fluorescence polarization assays may further be prone to problems relating to scattering-induced depolarization when the solution is turbid.
Standard microtiter plates have a base of approximately 128 mm×86 mm in versions with 96, 384 or 1,536 wells. A known automated standard method is the analysis of a microtiter plate with the aid of commercial microtiter plate readers having a fixed geometry for measuring excitation and emission of the fluorescent dye. For an extensive and complete analysis, the microtiter plate is transported electromechanically and sequentially, well-by-well, into the excitation/measurement position. There exist measuring systems with different illuminating geometries for perpendicular excitation from above or below and measuring the fluorescence from above or, through the transparent base of microtiter plates, from below, as is described, for example, in DE 197 20 667 A1 and corresponding U.S. Pat. No. 5,933,232. Since high-throughput screening for drug research requires several millions of chemical substances to be tested for their action in as short a time as possible, a high rate of measurement is a prerequisite for this high throughput. In the case of conventional fluorescence readers, moving the plate mechanically stands in the way of this. The measurement times for a microtiter plate with 1,536 wells which have to be addressed individually are in the minute range.
U.S. Pat. No. 5,096,807 discloses an image-based or imaging immunoassay detection apparatus system and method capable of imaging multiple light emitting reactions from small volume samples simultaneously and quantifying the same. Although imaging is obviously advantageous with regard to the prerequisite for high throughput, the illumination and detection geometry of conventional fluorescence-measuring systems, whether sequential or simultaneous using imaging methods, in which the sample is excited perpendicularly from below or from above and the fluorescence is detected likewise perpendicularly from below or from above, proves to be very disadvantageous for both adherent cell assays and homogeneous fluorescence assays for the reasons described above. Furthermore, because the path of the excitation light and emission light is unitary in the geometry of conventional fluorescence-measuring systems, transmission or reflection of the excitation light into the optical path of fluorescence detection may further limit quantitation of fluorescence measurements due to background caused by spectral leakage, or otherwise cause high expense with regard to spectral filtering technology.
The problem of interfering background fluorescence for adherent cell assays is addressed in U.S. Pat. No. 6,420,183, where an absorption dye is added to the supernatant solution to eliminate the exciting beam and the emitted radiation in the supernatant liquid over the cell layer to be observed. However, the use of absorbent dyes is also problematic because, on the one hand, their biochemical reaction is unclear and, on the other hand, the absorption in the supernatant liquid is incomplete and can ultimately also have an undesirable effect in the cell layer.
U.S. Pat. No. 5,355,215 discloses an instrument that specifically reduces the unwanted background fluorescence of the supernatant liquid and accordingly improves the wanted signal from a cell layer at the transparent base of the wells. The excitation light impinges on the base of the microtiter plate at an oblique incident angle from below and, in addition, the excitation light bundle cross section per well is limited through a multi-pinhole diaphragm in order to observe the fluorescent radiation, as far as possible, only from a small section volume at the base of each well. As a general condition, an optical illumination axis or detection axis is directed at an angle to the normal direction of the microplate. However, due to divergence of the excitation illumination, the excited liquid volume in the wells is dependent upon position.
FIGS. 2A, 2B, and 3 show an example of position dependence of the excited liquid volume. FIGS. 2A and 2B show microtiter plate 130 in cross-section. Excitation radiation path 110 is divergent and illuminates the fluorescent sample substances in wells A-H. FIG. 3 shows the relative variation of the excited volume of the fluorescent sample substances within each well, with the greatest excited volume corresponding to well H, the well closest to the radiation source. The detected fluorescence signal is typically proportional to the excited volume, so the variation in the excited volume directly corresponds to a variation in the detected fluorescence signal. This analysis assumes that the volumes of the substances within the wells are sufficiently large so that any variation in the volume or surface tension is inconsequential.
U.S. Pat. No. 6,985,225 discloses a fluorescence measurement system wherein the arrangement for fluorescence excitation contains a two-dimensionally extended sample-receiving device and at least two illumination sources for exciting the fluorescence of the samples. The illumination sources are extended linearly and arranged in such a way that the illuminated area of the sample-receiving device is, as far as possible, homogeneously illuminated directly or via deflecting mirrors at an opening angle of ≦30°. A detector system for the fluorescence light from the sample-receiving device is arranged on either side of the sample-receiving device in such a way that it detects fluorescence emission from the area of measurement at an angle outside the range of reflection of the excitation light of the illumination sources at the illuminated area of the sample-receiving device, preferably at an angle in the range from 80° to 100°, particularly preferably about 90°, to the extended plane of the area of the sample-receiving device. However, due to divergence of the illumination, the excited liquid volume in the wells is dependent upon position. Furthermore, the fluorescence detection volume is not limited to a small section at the base of the sample container, for example a tissue culture dish or each well of a microtiter plate.
FIGS. 4 and 5 show an example of position dependence of the excited liquid volume for the case of two excitation radiation sources. FIG. 4 shows microtiter plate 130 in cross-section. Excitation radiation paths 110 and 111 are divergent and illuminate the fluorescent sample substances in wells A-H. FIG. 5 shows the relative variation of the excited volume of the fluorescent sample substances within each well, with the greatest excited volumes corresponding to wells A and H, the wells closest to the radiation sources. The detected fluorescence signal is typically proportional to the excited volume, so the variation in the excited volume directly corresponds to a variation in the detected fluorescence signal. This analysis assumes that the volumes of the substances within the wells are sufficiently large so that any variation in the volume or surface tension is inconsequential.
U.S. Pat. No. 7,199,377 discloses a device for optical analytic measurement in a multisample carrier, wherein, during excitation of all of the wells, the fluorescence radiation of each well is measured simultaneously without, as far as possible, impermissible contributions of background radiation which falsify the characteristic emission of the sample material. The excitation light is directed from a light source on the multisample carrier coaxially in a ring-shaped manner around an optical axis wherein the optical axis is oriented in direction of a surface normal of the multisample carrier and coaxial to the direction of the readout beam path. A ring mirror unit with at least one curved ring mirror is arranged coaxial to the optical axis in such a way that the excitation light illuminates the multisample carrier homogeneously, as far as possible, on all sides at an oblique incident angle. However, due to divergence of the excitation illumination, the excited liquid volume in the wells is dependent upon position. Furthermore, the fluorescence detection volume is not limited to a small section at the base of the sample container, for example a tissue culture dish or each well of a microtiter plate.
US Publication 2003/0010930 discloses an arrangement for reading out the fluorescence radiation of specimen carriers with a plurality of individual specimens. For purposes of exciting fluorescence radiation in selected individual specimens, a switchable electro-optical matrix is provided for generating illumination which is limited in a spatially defined manner. An arrangement is disclosed for reading out the fluorescence radiation of selected individual specimens of multispecimen carriers having a switchable electro-optical matrix for generating illumination which is limited in a spatially defined manner. An optical system images the electro-optical matrix on the specimen carrier, and a high-sensitivity photoreceiver provides integral measurement of the fluorescence radiation of the excited individual specimens of the specimen carrier. A spatially differentiated illumination of a specimen carrier with a plurality of specimens is disclosed using an electro-optical matrix which minimizes the proportion of excitation radiation contributing to the fluorescence signal in high-resolution imaging. The electro-optical matrix and the specimen carrier are inclined relative to the optical axis of the optical system and are subject to a Scheimpflug condition. The angles of inclination of the electro-optical matrix and of the specimen carrier are selected such that the excitation radiation imaged by the light source unit on the specimen carrier is reflected in such a way that essentially no excitation radiation reaches the detection beam path. However, the fluorescence measurement volume is not limited to a small section at the base of the sample container, for example a tissue culture dish or each well of a microtiter plate.
Hence, there remains a need for an improved apparatus and method for fluorescence measurements of substances in sample carriers, such as tissue culture dishes and microtiter plates, as typically used for adherent cell assays and homogeneous fluorescence assays which solves the problems of the previously discussed known systems. Such an improved apparatus and method desirably would be capable of:
imaging-based fluorescence measurement, as required for simultaneous measurement of fluorescence from a plurality of sample volumes as desirable for high throughput;
wide-field excitation, as required for imaging-based fluorescence measurement;
angular separation of the optical paths of incident and reflected excitation light from the optical path of fluorescence measurement, as desirable to suppress spectral leakage and/or minimize cost of spectral filtration; and
limitation of the fluorescence measurement to a small volume selected at a depth in the sample carrier proximate to the optically transparent bottom member of the carrier, in order to render the fluorescence measurements immune to variability in total volume of solution and/or surface tension, to make the position of the fluorescence measurements insensitive to divergence of the excitation illumination, and to minimize the effects of fluorescent or autofluorescent supernatant, attenuation of both the excitation and emission radiation in the solution, and scattering-induced depolarization when the solution is turbid. It would further be desirable if only one excitation path were used to minimize cost and complexity of the apparatus.